The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.
CD164 is a member of the mucin-like receptor or sialomucin superfamily of glycoproteins. Sialomucins are transmembrane glycoproteins ranging from 50-3000 kD exhibiting limited similarity at the cDNA and amino acid levels. Mucin-like expressed proteins share the common characteristic of bearing numerous O-glycosylations linked to serine and threonine residues, which infer multiple kinds of cell-cell or cell-extracellular matrix interactions. The dense array of O-linked side chains are characterized by an extended structure that makes many of the mucin-like molecules long enough to protrude beyond the polysaccharide glycocalyx that surrounds the cell and also by the optimal exposure and high multiplicity of the terminal sugars. By virtue of the structural configuration as well as negative charge, mucin-like glycoproteins may act as a repulsive barrier unless cells bear specific receptors for mucin (adhesion). Functions of mucin receptors depend on cell types and states of activation correlated with the core mucin peptide and with the cell-specific expression of glycosyl transferases, which in turn regulate the structure and presentation of the O-linked oligosaccharide sidechains, membrane anchorage, signal transduction abilities and or/the trafficking of the mucin to the correct cellular domain.
Human CD164 is an ortholog of murine MGC-24v (M. musculus) and rat endolyn (R. norvegicus), a membrane protein found in lysosomal and endosomal compartment of mammalian cells. The relationships amongst different isoforms, together with functionally important domains and subcellular distribution of CD164/endolyn, have been described (Chan Y H et al., J. Biol. Chem., 276: 2139-2152, 2001).
In its native state, human CD164 is a disulphide-linked homodimer of two 80-85 kDa subunits. CD164 is highly glycosylated, containing both O- and N-linked glycans. The extracellular region is comprised of two mucin domains (I and II) linked by a non-mucin domain containing intra-disulphide bridges as well as a cysteine-rich motif that resembles a consensus pattern previously found in growth factor and cytokine receptors. CD164 also contains a single-pass transmembrane domain and a 13-amino acid intracellular region that include a C-terminal motif (i.e. YHTL) able to target the protein to endosomes and lysosomes.
Four human CD164 mRNA species have been described arising by alternative splicing of six bona fide exons from a single genomic transcription unit located on human chromosome 6q21 (Zannettino A, J Biol Regul Homeost Agents, 15: 394-396, 2001; Watt and Chan, Leuk Lymph, 37(: 1-25. 2000). There are probably 4 alternative promoters, two non-overlapping alternative last exons and one internal intron which is not always spliced out. The predominant CD164 (E1-6) isoform represents a 178 amino acid type I transmembrane glycoprotein. The other described isoforms are a sialomucin CD164 or CD164 isoform delta 5 containing 178 amino acids; a 184 residues CD164 isoform delta 4; and a 200 kD principally soluble isoform termed MGC-24 (for Multi-Glycosylated Core protein of 24 kD) lacking the transmembrane anchoring motif and having 189 residues. All isoforms are highly glycosylated proteins with O- and N-linked glycosylation sites (FIG. 1).
CD164 functions include mediating, or regulating, haematopoietic progenitor cell adhesion and the negative regulation of their growth and/or differentiation. CD164 is usually expressed by CD34+ and CD34 lo/− haematopoietic stem cells and associated microenvironmental cells (Watt et al., Blood, 92: 849-866, 1998). CD164 is also expressed by committed myeloid and erythroid colony forming cells, on bone marrow stromal and endothelial cells, weakly on lymphocytes, and on mesenchymal stem cells. CD164 may play a key role in haematopoisesis by facilitating the adhesion of human CD34+ cells to bone marrow stroma and by suppressing CD34+CD38 lo/− haematopoietic progenitor cell proliferation, acting as a potent signaling molecule (Zannettino et al. Blood, 92: 2613-2628, 1998).
These effects involve the CD164 class I and/or II epitopes recognized by the monoclonal antibodies (mAbs) 105A5 and 103B2/9E10. The epitopes are carbohydrate-dependent and are located on the N-terminal mucin domain I (Watt et al., Blood, 95, 3113-3124, 2000; Doyonnas et al., J Immunol, 165: 840-851, 2000). The interaction of haemotopoietic cells with stromal/endothelial cells in their immediate microenvironment is thought to be of major importance in the regulation of haematopoietic stem self-renewal, quiescence, commitment and migration. These interactions involve cooperation between adhesion receptors, their cognate ligands and cytokines. A range of cell adhesion molecules (CAMS) including the Ig, integrin, cadherin, selectin and mucin-like protein families, participate in these processes.
In vitro, CD164 showed a role in myogenic differentiation (Lee et al., Mol Cell Biol, 21: 7696-7706, 2001). Overexpression of CD164 in myoblast cell lines accelerated expression of biochemical markers of differentiation and enhanced formation of multinucleate myotubes, whereas antisense CD164 or soluble extracellular regions of CD164 inhibited myogenesis.
The peanut agglutinin (PNA)-binding site of soluble MGC-24 represents a tumor associated carbohydrate marker expressed in many carcinomas. Total MGC-24 mRNA was found to be lower in human colorectal carcinomas as compared with normal adjacent mucosal tissues (Matsui et al., J Biochem, 127: 1103-1107, 2000). Lymphatic vessel invasion by the carcinoma was correlated to low levels of MGC-24 mRNA in colon carcinomas, whereas high levels did correlate with less venous invasion and less remote metastasis. Monoclonal antibodies specific for CD164 could prove useful for cancer diagnosis or therapy and haematopoiesis inhibition (EP889054, EP761814).
Other CD164-like proteins have been disclosed (NOV25, WO 02/098917; SEQ ID NO: 7852, EP1033401; FIG. 1), but their biological properties have not been analyzed.